Cholesterol and glycolipids self-associate in lipid bilayers to form organized compositional microdomains (Thompson, T. E., et al., Annu. Rev. Biophys. Chem. 14:361 (1985)). Glycosyl-phosphatidylinositol (GPI)-anchored proteins and other lipid-linked proteins may preferentially partition into glycolipid microdomains that are resistant to nonionic detergent solubilization (Schroeder, R., et al, Proc. Natl. Acad. Sci. USA 91:12130 (1994); Brown, D. A. and Rose, J. K., Cell 68:533 (1992); Letarte-Murhead, M. et al., Biochem. J 143:51 (1974); Hoessli, D. and Runger-Brandle, E., Exp. Cell. Res. 166:239 (1985); Hooper, N. M. and Turner, A. J., Biochem. J. 250:865 (1968); Sargiacomo, M. et al., J. Cell. Biol. 122:789 (1993); Lisanti, M. P. et al., J. Cell. Biol. 123:595 (1993)). GPI-anchored proteins appear to be sorted into glycolipid, detergent-resistant xe2x80x9craftsxe2x80x9d in the trans-Golgi network for polarized delivery to the cell surface by smooth exocytotic carrier vesicles which are resistant to detergents and also contain caveolin (Brown, D. A. and Rose, J. K., Cell 68:533 (1992); Sargiacomo, M. et al, J. Cell. Biol. 122:789 (1993); Lisanti, M. P. et al., J. Cell. Biol. 123:595 (1993); Brown, D. et al, Science 245:1499 (1989); Simons, K. and van Meer, G., Biochemistry 27:6197 (1988); Garcia, M. et al., J. Cell Sci 104:1281 (1993); Kurzchalia, T. V. et al., J.Cell Biol. 118:1003 (1992); Dupree, P. et al., EMBO J. 12:1597 (1993); Hannan, L. A. et al., J. Cell. Biol. 120:353 (1993)). Caveolae are smooth membrane invaginations that are also resistant to detergent extraction; they exist on the surface of many different cell types, and are especially abundant in endothelium. Caveolae are apparently also rich in glycolipids, cholesterol, and caveolin (Kurzchalia, T. V. et al., J. Cell Biol. 118:1003 (1992); Schnitzer, J. E. et al., Proc. Natl. Acad. Sci. USA 92:1759 (1995)). Low-density, Triton-insoluble membranes are frequently equated with caveolae (Sargiacomo, M., et al., J. Cell Biol. 122:789 (1993); Lisanti, M. P. et al., J. Cell. Biol. 123:595 (1993); Chang, W.-J. et al., J. Cell. Biol. 126:127 (1994); Lisanti, M. P. et al., J. Cell. Biol. 126:111 (1994)), but recent work has shown they have a mixture of detergent-resistant microdomains (Schnitzer, J. E. et al., Science 269:1435-1439 (1995)). Characterization of caveolae shows that they are very enriched in caveolin; the glycolipid GM1; the plasmalemmal CA2+-dependent adenosine triphosphatase; and the inositol 1,4,5-triphosphate receptor; these four molecules have all been shown by independent means to reside on the cell surface almost exclusively in caveolae (Dupree, P., et al., EMBO J. 12:1597 (1993); Parton, R. G., J. Histochem. Cytochem. 42:155 (1994); Rothberg, K. G. et al., Cell 68:673 (1992); Montessano, R. et al., Nature 296:651 (1982); Fujimoto, T., J. Cell. Biol. 120:1147 91993)) and thus represent key markers of caveolae.
Caveolae have been implicated not only in signaling but also in transport via endocytosis, transcytosis, and potocytosis (Montessano, R. et al., Nature 296:651 (1982); Schnitzer, J. E., Trends Cardiovasc. Med. 3:124 (1993); Oh, P. et al., J. Cell Biol. 127:1217 (1994); Schnitzer, J. E. and Oh, P., J. Biol. Chem. 269:6072 (1994); Schnitzer, J. E. et al., Proc. Natl. Acad. Sci. USA 92:1759-1763 (1995); Schnitzer, J. E. et al., J. Biol. Chem. 270:14399-14404 (1995); Millci, A. J. et al., J. Cell Biol. 105:2604 (1987); Anderson, R. G. W. et al., Science 265:410 (1992)). However, there is disagreement as to whether caveolae serve as signaling centers (see Liu, J. et al., J. Biol. Chem 272:7211-7222 (1997), Schnitzer, J. E. et al., Mol. Biol. Cell 5:75a (1994); Schnitzer, J. E. et al., Proc. Natl. Acad. Sci. USA 92:1759-1763 (1995); Schnitzer, J. E. et al., J. Biol. Chem. 270:14399-14404 (1995); contrast with Stan, R.-V. et al., Mol. Biol. Cell 8:595-605 (1997)). The exact physiological composition and functions of caveolae remain undefined.
The present invention is drawn to methods of producing purified caveolae, as well as the purified caveolae produced by the methods, and uses of the purified caveolae. In the methods, immunoisolation of caveolae is performed, using an antibody that is specific for caveolin and that is able to bind oligomerized caveolin found around intact caveolae. Immunoisolation can be performed on a wide variety of starting materials, including cells of interest, such as cultured cells or cells isolated from a tissue; a tissue itself; cell lysate; microsomes derived from cells or from tissue; or a sample of plasma membranes.
In one embodiment, the starting material can be subjected to membrane disruption method and/or a preliminary separation step prior to the immunoisolation. If such a separation step is performed, the separation is based on a physical characteristic of cell membranes (for example, density, size, or phase separation), in order to provide a starting material containing a concentrated amount of plasma membranes. The initial separated fractions (e.g., the lowest density fractions in a density separation) are then collected, and subjected to the immunoisolation method to separate caveolae from other materials in the initial fractions.
During the immunoisolation, a sample of interest (e.g., the starting material, sample of plasma membranes, or initial fractions) that comprises plasma membranes is incubated, preferably for a brief time period (e.g., for less than approximately 2 hours, preferably for approximately one hour or less), with the antibody that is specific for caveolin. Caveolae that are bound to the antibody are then separated from other materials in the sample of interest, thereby producing purified caveolae.
In a preferred embodiment, a sample of plasma membranes from cells of interest is used as the sample of interest. The sample of plasma membranes can be subjected to a membrane disruption method, such as sonication or shearing, to produce disrupted plasma membranes. If desired, the disrupted plasma membranes can then be subjected to separation based on a physical characteristic of the membrane, as described above. The resultant material is then subjected to the immunoisolation method to separate caveolae from other materials in the initial fractions.
The methods of the invention provide simplified, efficient means to produce purified caveolae, while minimizing contamination and avoiding loss of molecules that dissociated from caveolae with time. The caveolae produced by the methods more closely resemble caveolae in their native state; as the methods eliminate extended immunoisolation methods which would otherwise result in significant loss of various caveolae components as well as adsorption of nonspecific proteins or contaminating membranes. The methods can be used to produce purified caveolae from a wide variety of cells or tissues, including not only endothelial cells and tissues, but also other (non-endothelial) cells and tissues, as well as cultured cells.